Methods and compositions for reducing oxidative stress in an animal

ABSTRACT

Methods for the reduction or prevention of oxidative stress in an animal comprising administering to the animal an effective amount of a composition comprising astaxanthin and/or Vitamin E are described. Also described are compositions comprising astaxanthin and/or Vitamin E, the compositions being effective for the reduction or prevention of oxidative stress in an animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This claims benefit of U.S. Provisional Application No. 60/562,815,filed Apr. 16, 2004, the entire contents of which are incorporated byreference herein. This also is related to U.S. application Ser. No. [notyet assigned] filed concurrently herewith. The entire contents of thatapplication are also incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to methods and compositions effectivein reducing and/or preventing oxidative stress in an animal byastaxanthin and/or Vitamin E supplementation.

BACKGROUND OF THE INVENTION

Cells obtain energy from the oxidation of a variety of organicmolecules, and oxygen is the primary oxidant in the biochemicalreactions that perform this function. Oxidative stress, which resultsfrom the metabolic reactions that use oxygen, may be considered adisturbance in the equilibrium status of pro-oxidant/anti-oxidantsystems in intact cells. Cells have intact pro-oxidant/anti-oxidantsystems that continuously generate and detoxify oxidants during normalaerobic metabolism. When additional oxidative events take place, thepro-oxidant systems outbalance those of the anti-oxidant, which mayresult in oxidative damage to cell components including lipids,proteins, carbohydrates, and nucleic acids. Mild, chronic oxidativestress may alter the anti-oxidant systems by inducing or repressingproteins that participate in these systems, and by depleting cellularstores of anti-oxidant materials such as glutathione and Vitamin E.Severe oxidative stress may ultimately lead to cell death.

An imbalance in pro-oxidant/anti-oxidant systems may result from anumber of different oxidative challenges, including radiation,metabolism of environmental pollutants and administered drugs, as wellas immune system response to disease or infection. The immune responseis of particular significance since many toxic oxidative materials aregenerated in order to attack invading organisms. A variety of chemicalscalled radicals have roles in these processes. A radical species is anyatom that contains one or more orbital electrons with unpaired spinstates. A radical may be a small gas molecule such as oxygen or nitricoxide, or it may be a part of a large biomolecule such as a protein,carbohydrate, lipid, or nucleic acid. Some radical species are veryreactive with other biomolecules and others, like the normal tripletstate of molecular oxygen, are relatively inert.

Of interest with respect to oxidative stress are the reactions ofpartially reduced oxygen products and radical and non-radical speciesderived from them. A variety of reactive nitrogen species derived fromthe reactions of nitric oxide also play important roles in oxidativestress.

Oxidative stress has been implicated in human and animal disease. Cellshave, however, multiple protective mechanisms against oxidative stressthat act in preventing cell damage. Many dietary constituents areimportant sources of protective agents including anti-oxidant vitaminsand minerals as well as food additives that might enhance the action ofnatural anti-oxidants. The effectiveness of an anti-oxidant in oxidativestress may be dependent on the specific molecules causing the stress,and the cellular or extracellular location of the source of thesemolecules.

Intense exercise can contribute significantly to oxidative stress in anumber of ways. Most individuals have at some time in their livesexperienced soreness and fatigue after physical exertion. For animalsthat undergo intense, frequent exercising, the effects of oxidativestress can have negative effects on performance.

Intense exercise results in a number of physiological changes in thebody. First, aerobic respiration is dramatically increased, therebyincreasing superoxide anion generation as much as 10-fold or more(Halliwell, B. (1994) Free radicals and antioxidants: a personal view.Nutr. Rev. 52:253-265), in addition to increasing exposure toenvironmental oxidative insults such as air pollution. Second, muscleand joint inflammation often result from intense exercise, thustriggering tissue infiltration of neutrophils and subsequent release ofreactive oxygen species during the “oxidative burst” characteristic ofactivated neutrophils mediated by the immune response.

Enhanced antioxidant intake in humans has been reported to decrease therisk of developing specific forms of cancer and to enhance immunefunction. The effects of dietary antioxidant intake on pathological andphysiological processes such as the aging process and exercise in dogshave been reported in the scientific literature. The effects of enhancedintakes of Vitamins E and C on immune function, free radical formationand free radical scrubbers have also been reported.

Cataracts may develop due to metabolic disorders such as diabetes, andfrom exposure to light, followed by subsequent oxidation of the lens.Oxidative stress, therefore, directly contributes to cataract formation.Primary (diene conjugates, cetodienes) lipid peroxidation (LPO) productsaccumulate during the initial stages of cataract formation while LPOfluorescent end-products are dominant in the later stages (Babizhayev etal. (2004) Drugs R. D. 5(3):125-139). Lens opacity correlates with theLPO fluorescent end-product accumulation in the tissue, and decreasedreduced glutathione leads to sulfhydryl group oxidation of lens proteins(Babizhayev et al. (2004) Drugs R. D. 5(3):125-139). It has been shownthat direct injection of LPO products into the vitreous induces cataractformation (Babizhayev et al. (2004) Drugs R. D. 5(3):125-139). Thus,peroxide damage of lens fiber membranes appears to initiate thedevelopment of cataracts (Babizhayev et al. (2004) Drugs R. D. 5(3):125-139).

Osteoarthritis (OA) is also related to oxidative stress. Free radicals,including nitric oxide, superoxide anion and hydrogen peroxide, lead toupregulation of enzymes responsible for damage to articular cartilage.These enzymes (MMPs) are specific for collagen, elastin and gelatin.

Oxygen radicals with unpaired electrons are produced as a normal part ofoxygen metabolism. These reactive molecules may cause damage to, forexample, proteins, nucleic acids (i.e., DNA) and/or membranes which mayresult in serious cell injury and disease in the whole animal. Thisprocess has been associated with the aging process, degenerativediseases, and cancer.

Animals that may be particularly vulnerable to oxidative damage orstress include those that are very active. For instance,well-conditioned canine athletes are healthy animals, which by virtue oftheir tremendous rates of oxygen consumption generate more free radicalseach day than their more sedentary counterparts. The changes in immunefunction, metabolic intermediates, tissue stores of antioxidants andantioxidant enzymes induced by free radical production are greater incanine athletes than sedentary animals.

Previous studies examining hard working dogs revealed that exercise wasassociated with a significant increase in plasma concentrations ofisoprostanes, a stable by product of lipid peroxidation. These samestudies also demonstrated a decrease in plasma Vitamin E concentrationsduring exercise. Further studies examined the effect of Vitamin Esupplementation on these parameters. Vitamin E supplementation helpeddecrease or alleviate the exercise associated drop in plasma Vitamin Econcentration but did not decrease the elevations in plasmaisoprostanes.

Astaxanthin is a mixed carotenoid which may be found in a number ofsources; it is present in high levels in algae. This pigment protectsthe algal organism from damage due to ultraviolet radiation exposure.Several studies have demonstrated immuno-stimulatory properties ofastaxanthin in cultured cells as well as whole animals (mice). It isalso used in sunscreen lotions as it has been demonstrated to diminishreddening of the skin after sun exposure. Astaxanthin supplementationhas also been associated with increased endurance in untrained humansubjects.

SUMMARY OF THE INVENTION

In certain aspects, the present invention relates to methods for thereduction or prevention of oxidative stress in an animal comprisingadministering to the animal an effective amount of a compositioncomprising astaxanthin and/or Vitamin E. Additional aspects of thepresent invention relate to compositions comprising astaxanthin and/orVitamin E, the compositions being effective for the reduction orprevention of oxidative stress in an animal.

In one aspect, the invention provides compositions comprisingastaxanthin wherein the composition is adapted for use by a companionanimal. In various embodiments, the compositions are formulated for usein a reducing oxidative stress in an animal, and comprise astaxanthinand Vitamin E.

In another aspect, the invention provides methods for attenuatinginflammation, enhancing immunity, enhancing longevity, and combinationsthereof, comprising administering to a companion animal a compositioncomprising an effective amount of astaxanthin.

In another aspect, the invention provides methods of reducing oxidativestress in an animal by administering a composition of the invention. Theinvention further provides methods for reducing at least one oxidativestress-associated molecule, such as, but not limited to nitric oxide,malondialdehyde, F2 isoprostanes, lipid peroxidation products, creatinekinase, superoxide anion and hydrogen peroxide.

In another aspect, the invention provides a method of promoting exerciserecovery in an animal comprising administering to the animal aneffective amount of a composition comprising astaxanthin effective topromote exercise recovery.

Other features and advantages of the present invention will beunderstood by reference to the detailed description and the examplesthat follow.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Before describing the invention in detail, it should be understood thatthis invention is not limited to the particularly exemplified systems orprocess parameters as described in the specification for theseparameters may of course vary. It is to be further understood that theterminology used herein is for the purpose of describing particularembodiments of the invention only, and is not intended to limit thescope of the invention in any manner for the invention in any manner.

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

“Effective amount” refers to an amount of a compound, material,composition, and/or dosage form as described herein that may beeffective to achieve a particular biological result. Such results mayinclude, but are not limited to, reduction and/or prevention ofoxidative stress. Such effective activity may be achieved, for example,by causing the ingestion of compositions according to aspects of thepresent invention.

“Mammal” refers to any of a class of warm-blooded higher vertebratesthat nourish their young with milk secreted by mammary glands and haveskin usually more or less covered with hair, and non-exclusivelyincludes cats and dogs.

“Oxidative stress” refers to the condition characterized by an excess ofoxidants and/or a decrease in antioxidant levels. Cellular oxidants mayinclude, but are not limited to, one or more of: radicals of oxygen(superoxide anion, hydroxyl radical, and/or peroxy radicals); reactivenon-radical oxygen species such as, for example, hydrogen peroxide andsinglet oxygen; carbon radicals; nitrogen radicals; and sulfur radicals.The condition of oxidative stress may result in, for example, cellulardamage, impaired performance of cells and/or cell death.

It must be further noted that as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include theplural referents unless the context clearly dictates otherwise.

In addition, the term “about” as used herein is intended to indicate arange of values of 10% greater and lesser than the indicated value.Thus, about 5% is intended to encompass a range of values from 4.5% to5.5%.

Unless defined otherwise, all technical and scientific terms have thesame meaning as commonly understood by one of ordinary skill in the artto which the invention pertains. Although a number of methods andmaterials similar or equivalent to those described herein can be used inthe practice of the present invention, the preferred materials andmethods are described herein.

All numerical ranges described herein include all combinations andsubcombinations of ranges and specific integers encompassed therein.

According to certain aspects of this invention, exercise was utilized asa model to increase free radical production in an animal.

The present invention relates to any animal, preferably a mammal; morepreferably to cats, and most preferably to dogs.

As used herein, “Vitamin E” refers to any of a group of relatedcompounds with similar biological antioxidant activity, includingalpha-tocopherol but also including other isomers of tocopherol and therelated compound tocotrienol. The molecule is lipophillic and may residein cell membranes. According to certain aspects of the invention,alpha-tocopherol may be a preferred form.

As used herein, “astaxanthin” refers to a mixed carotenoid occurringnaturally in a wide variety of living organisms; it may also be producedsynthetically. Sources of astaxanthin include, but are not limited to,crustaceans, including shrimp, crawfish, crabs and lobster; fish,including, for example, salmon; the pink yeast Xanthophyllomyces ; andthe microalga Haematococcus pluvialis . In certain preferred embodimentsof the invention, the source of astaxanthin is NatuRose®, available fromCyanotech (Kailua-Kona, HI).

As used herein, a “foodstuff” refers to any substance that can be usedor prepared for use as food. As used herein, a “food” is a materialconsisting essentially of protein, carbohydrate and/or fat, which isused in the body of an organism to sustain growth, repair and vitalprocesses and to furnish energy. Foods may also contain supplementarysubstances such as minerals, vitamins and condiments. The term foodincludes a beverage adapted for human or animal consumption. As usedherein, a “pharmaceutical” is a medicinal drug. A pharmaceutical mayalso be referred to as a medicament. As used herein, a “dietarysupplement” is a product that is intended to supplement the diet; it maybear or contain any one or any combination of the following dietaryingredients: a vitamin, a mineral, an herb or other botanical, an aminoacid, a dietary substance for use to supplement the diet by increasingthe total daily intake (including, without limitation, enzymes ortissues from organs or glands), a concentrate, metabolite, constituent,or extract.

According to an embodiment of the present invention, a method isprovided comprising the administration of astaxanthin and/or Vitamin Eto an animal, the method being effective for the reduction and/orprevention of oxidative stress in the animal. According to an aspect ofthe invention, the method comprises the administration of an effectiveamount of astaxanthin. According to another aspect of the invention, themethod comprises the administration of both astaxanthin and Vitamin E.Preferably, the astaxanthin and/or Vitamin E may be administered to ananimal in a diet, food, foodstuff, dietary supplement, or pharmaceuticalcomposition as, for example, described herein.

According to a preferred embodiment of the invention, the method iseffective for facilitating oxidative stress recovery in animals.According to a particularly preferred embodiment, the method iseffective for facilitating recovery from oxidative stress due toexercise.

The methods and compositions according to certain aspects of theinvention may possess anti-aging effects in animals and therefore may bebeneficial in promoting longevity of an animal. The compositions andmethods of the invention are also useful in reversing, preventing orreducing detrimental conditions associated with oxidative stress, suchas, but not limited to osteoarthritis, cataract formation, weak immunesystems, trauma and infection. The compositions and methods are furtheruseful in enhancing normal immune system function.

Without being limited by any theories or any particular modes of actionof the invention, it is believed that the two antioxidants astaxanthinand Vitamin E may have differing efficacies against different types ofoxidative stress. Vitamin E may be most effective against membrane lipidoxidation associated with exercise-induced elevations of oxygenmetabolism. Astaxanthin may be most effective against oxidative stresscaused by increased immune cell activity in the 24 hours followingexercise. These observations suggest a potential for at least additiveeffects, and possibly synergism, if the two antioxidants are combined.

To illustrate certain aspects of the invention, Alaskan sled dogs ran 30miles for three consecutive days and biomarkers, such as F-2isoprostanes, were measured pre-, post-, 24hr, and 48 hr after exercise.Because the rate of free radical generation in these exercising animalsis greater than in the average household pet, the effect of years offree radical exposure was simulated in a short period of time: days toweeks. The blood analysis from these exercising dogs demonstratedsignificant declines in plasma Vitamin E and ceruloplasmin along withsignificant increases in isoprostanes, creatine kinase (CK) and uricacid (UA). When prostaglandins are exposed to free radicals they areconverted into isoprostanes, which are stable for several hours andeasily measured by a commercially available ELISA kit. Further, it hasbeen shown that immune cell activity increases the concentration oflipid oxidation markers after exercise.

Three groups of twelve dogs (mixed breed Alaskan sled dogs (huskypointer crosses)) were fed commercially available Pro Plan® Performancechicken and rice formula dog food as a basal diet, and each group wasfed either: a placebo consisting of about 0.5 g maltodextrin, about 2 mgastaxanthin, or about 400 mg Vitamin E (alpha-tocopherol). Pro Plan® dogfood is available from Nestle Purina Pet Care Company in St. Louis,Missouri. The dogs were trained three times a week on an exercise wheelfor a period of six weeks. On the sixth week, the dogs rested for fourdays and then exercised three days in a row. Blood samples werecollected before exercise on day one, immediately after exercise on daythree, and at 24 hours and 48 hours after exercise. Plasma from theblood samples was analyzed for concentrations of malondialdehyde, F-2isoprostanes, Vitamin E, creatine kinase activity as well as oxygenradical absorption capacity (ORAC).

The effects of supplementing Vitamin E, astaxanthin, or a placebo onseveral indices of lipid oxidation and immune function were examined.Vitamin E was chosen because it is a common antioxidant and provided abenchmark upon which to compare other antioxidants. Astaxanthin is amixed carotenoid which may be obtained from a number of sources,including, without limitation, being harvested from microalgae. It issoluble in both lipid and water compartments of the cell and thereforehas a broader distribution than either Vitamin E (lipid soluble only) orVitamin C (water soluble only). Astaxanthin has 4 times the in vitrooxygen radical absorptive capacity of Vitamin E. It is also reported tobe more potent than Vitamin E or Vitamin C in preventing lipidperoxidation and scavenging free radicals.

According to certain aspects of the invention, dietary intake patternsof antioxidants, such as Vitamin E and astaxanthin, are identified.These antioxidants yield optimal protection from free radical inducedchanges in exercising dogs. By measuring each antioxidant individuallyand then in combination with others any additive or synergistic effectsmay be observed and antioxidant systems that may protect both the cellmembrane and the cytosol may be developed.

According to an embodiment of the invention, a method of evaluating theeffect of a composition on oxidative stress in an animal is providedcomprising the steps of: a) administering the composition to the animal,b) measuring one or more indices of oxidative stress in the animal priorto training, c) training the animal according to an exercise regimen, d)measuring one or more indices of oxidative stress after training, e)comparing the differences in the indices of oxidative stress in b) andd) to similar differences in a control animal not administered thecomposition; wherein the composition is deemed to have an effect onoxidative stress if the result in e) differs between those of the animaltreated with the composition and those of the control animal.Preferably, the exercise regimen involves training an animal on anexercise wheel, a treadmill and/or a swimming pool. The use of anexercise wheel enables the investigator to standardize the exertion ofthe animal by employing one or more settings corresponding to a fixedspeed and distance. A treadmill may also enable such control andstandardization by allowing the investigator to set parameters such asspeed and inclination. A swimming regimen may also be standardized.Preferably, the indices of oxidative stress may be selected from one ormore of: plasma concentration of malondialdehyde, plasma concentrationof F-2 isoprostanes, plasma concentration of Vitamin E, creatine kinaseactivity, or oxygen radical absorption capacity (ORAC).

According to an embodiment of the present invention, a composition isprovided comprising astaxanthin and/or Vitamin E, the composition beingeffective for the reduction and/or prevention of oxidative stress inanimals. According to an aspect of the invention, the compositioncomprises an effective amount of astaxanthin. According to anotheraspect of the invention, the composition comprises astaxanthin andVitamin E.

According to a preferred embodiment of the invention, the composition iseffective for facilitating oxidative stress recovery in animals.According to a particularly preferred embodiment, the composition iseffective for facilitating recovery from oxidative stress due toexercise. In other embodiments, the composition is effective fortreating, delaying or preventing undesirable physiological conditionsassociated with oxidative stress. Examples include, but are not limitedto osteoarthritis, cataract formation, reduced or compromised immuneresponses, and increasing the robustness of normal immune responses.

According to certain aspects of the invention, a composition of theinvention may be useful as, for example, a diet, a food, a foodstuff, adietary supplement, or a veterinary therapeutical product. Thecompositions may optionally contain a carrier, a diluent, or anexcipient, chosen to be suitable for the intended use.

The compositions may be administered enterally, such as, for example, byan oral, intragastric, or transpyloric route. Many factors that maymodify the action of the composition can be taken into account by thoseskilled in the art; e.g., body weight, sex, diet, time ofadministration, route of administration, rate of excretion, condition ofthe subject, and reaction sensitivities and severities. Administrationcan be carried out continuously or periodically, such as once daily, oronce with every meal.

Certain aspects of the invention are preferably used in combination witha complete and balanced food (for example, as described in NationalResearch Council, 1985, Nutritional Requirements for Dogs, NationalAcademy Press, Washington D.C., or Association of American Feed ControlOfficials, Official Publication 1996). That is, compositions comprisingastaxanthin and/or Vitamin E according to certain aspects of thisinvention are preferably used with a high-quality commercial food. Asused herein, “high-quality commercial food” refers to a dietmanufactured to produce the digestibility of the key nutrients of 80% ormore, as set forth in, for example, the recommendations of the NationalResearch Council above for dogs. Similar high nutrient standards wouldbe used for other animals.

The dosages of the substance(s) used in various aspects of the presentinvention that will be most suitable will vary with the form ofadministration, the particular substance(s) chosen and the physiologicalcharacteristics of the particular animal receiving the dose. Accordingto certain aspects of the invention, preferred daily dosage ranges forVitamin E may be from about 15 to about 500 mg per animal per day, morepreferably from about 150 to about 450 mg per animal per day, morepreferably about 400 mg per animal per day. Preferred daily dosageranges for astaxanthin may be from about 0.001 to about 40 mg per animalper day. In some embodiments, the amount is from about 0.001 to about 10mg per animal per day. In other embodiments the amount is from about 1to about 40 mg per animal per day. In still other embodiments, theamount is from about 1 to about 20 mg per animal per day. In somepreferred embodiments, the amount is from about 0.1 to about 8 mg peranimal per day, more preferably about 2 mg per animal per day.

In some embodiments of the invention, the astaxanthin is admixed with afood product composition such that the composition comprises less thanabout 3% of astaxanthin by weight of the composition. In otherembodiments the composition comprises about 0.0001% to about 2% ofastaxanthin by weight of the composition. In other embodiments, thecomposition comprises about 0.001% to about 1% of astaxanthin by weightof the composition. In still other embodiments, the compositioncomprises about 0.001% to about 0.5% of astaxanthin by weight of thecomposition.

According to another aspect of the invention, there is provided adietary supplement useful for the reduction and/or prevention oxidativestress in an animal, the supplement comprising astaxanthin and/orVitamin E. The dietary supplement can be in any convenient form,including, without limitation, liquid, solid, or powder form. Solidforms of the supplement include, but are not limited to, a pill,biscuit, or treat.

According to certain embodiments of the invention, a dietary supplementcan be formed as a foodstuff with higher levels of astaxanthin and/orVitamin E which requires “dilution” before feeding to an animal. Thesupplement may be in any form, including, without limitation, solid(e.g. a powder), semi-solid (e.g. a food-like consistency/gel) or aliquid. The supplement may be administered to the animal in any suitablemanner. For example, the liquid form can conveniently be mixed in withfood or fed directly to the animal, such as, for example, via a spoon orvia a pipe-like device. In certain embodiments, the supplement can behigh in both components of astaxanthin and Vitamin E or can be acombined pack of two or more components, having the requiredconcentrations of astaxanthin and/or Vitamin E separately or in anysuitable combination.

EXAMPLES

The invention is further demonstrated in the following examples. Example1 is an actual example and Examples 2-6 are prophetic examples. Theexamples are for purposes of illustration and are not intended to limitthe scope of the present invention.

Example 1

Materials and Methods

All the dogs were maintained on the same basal diet (Purina Pro Plan®Performance chicken and rice formula) for one month prior to the onsetand the entire duration of the experiment. Each dog was fed as anindividual to maintain an optimal body condition score of 4/10. A bodycondition score of 4/10 indicates that the dogs were lean but notemaciated (1 is very thin, 10 is grossly obese).

Thirty-six sled dogs were housed at the Nestle Purina facility inSalcha, Alaska. The dogs were divided into three groups of 12 dogs sothat each group was alike in age, sex and ability distribution. Atreatment was randomly assigned to each group by drawing from a hat.Group “A” was assigned about 400 mg Vitamin E/day, group “B” wasassigned about 2 mg Astaxanthin/day and group “C” was assigned about500mg maltodextrin as a placebo. The people responsible for feeding,care, and training of the dogs were blind as to the identity oftreatments until the completion of the study. Dogs were treated for sixweeks before the onset of testing. They were also trained lightly fortwo sessions per week during this time period. Training sessionsconsisted of walking at 14 kilometers per hour for 2 hours in a 40-footdiameter circle tethered to a dog-walking wheel.

The testing took place over a six day period. On day one, pre-exerciseblood samples were drawn from all the dogs. On day two, the dogs rantwelve km while pulling an all terrain vehicle (ATV) weighing 500pounds. On day three, the dogs walked 16 miles on the dog wheel. On dayfour, the dogs again pulled the ATV for a distance of 12 kilometers.Immediately after exercise on day four, blood samples were drawn.Additional blood samples were drawn again at 24 hours and 48 hours afterexercise. This sampling schedule was designed to examine changes in theparameters measured during exercise and during recovery. Samples wereobtained by jugular venipuncture and collected into evacuated glasstubes containing sodium EDTA as an anticoagulant. Once collected theywere kept on ice for not more than 10 minutes before they werecentrifuged at 10,000 revolutions per minute (RPM) in 4°C. for a periodof 10 minutes. The plasma was transferred into freezer vials where thedead space was replaced with gaseous nitrogen to decrease furtheroxidation during storage. The vials were then capped and immediatelyquenched in liquid nitrogen. The vials were recovered from liquidnitrogen and stored at −70°C. until analyzed. Each plasma sample wasanalyzed for malondialdehyde concentration using a standard HPLC,F2-isoprostane concentration utilizing a mass spectrometer and standardELISA, Vitamin E concentration utilizing a standard HPLC, and OxidativeRadical Absorbance Capacity (ORAC test kit, Oxford Co.

F2 isoprostane and CK data was evaluated using a two way ANOVA forrepeated measures. Post hoc comparisons were made using a student's ttest corrected for repeated measures by the Bonneferroni method.Statistical significance was set at p<0.05 . The other data was alsoevaluated using ANOVA. The abbreviation “se” used with respect to thedata in the tables below refers to standard error.

Results

Plasma Vitamin E.

Table 1 shows the plasma values for Vitamin E. Plasma Vitamin Econcentrations were greatest for the Vitamin E treated group across alltime. periods. There was no significant difference between the plasmaVitamin E concentrations of Placebo and Astaxanthin (ATX) treated dogsfor any time period. The plasma Vitamin E concentrations of alltreatment groups tended to increase across time. All Vitamin Econcentrations were maintained well above the minimum of the normalrange for dogs established by the laboratory conducting the analyses.TABLE 1 Plasma Vitamin E (mean +/− se) Time Pre-ex Post-ex 24 hour post48 hour post Placebo 2170 1850 2380 2280 (+/−404) (+/−404) (+/−404)(+/−404) ATX 1635 1680 2000 1990 (+/−358) (+/−358) (+/−358)* (+/−358)Vitamin E 3370 3540 4720 4520 (+/−390)a (+/−390)*, a (+/−390)*, a(+/−390)*, a*= significantly different from pre exercise time period within thegroupa= Significantly different than other treatment groups within the timeperiod

Plasma Malondialdehyde (MDA).

Table 2 shows the values for plasma malondialdehyde. The only differencebetween the three treatment groups was observed at 24 hours postexercise where the Astaxanthin value was lower than either the placebovalue or the Vitamin E value. TABLE 2 Plasma MDA (mean +/− se) TimePre-ex Post-ex 24 hour post 48 hour post Placebo 1.13 1.46 1.4 1.41(+/−0.19) (+/−0.19) (+/−0.21) (+/−0.18) ATX 1.45 1.58 0.95 1.44(+/−0.24) (+/−0.41)  (+/−0.19)* (+/−0.19) Vitamin E 1.51 1.17 1.44 1.44(+/−0.17) (+/−0.26) (+/−0.40) (+/−0.10)*= Significantly different from pre-exercise and post-exercise valueswithin treatment groupa= Significantly different from placebo group within time period

Table 3 shows the change in MDA from baseline for each time period.Relative to the pre-exercise value all post-exercise samples wereelevated in the placebo group. There was no difference between thepre-exercise and post-exercise MDA values in the Astaxanthin group, butthe 24-hour post-exercise value was significantly lower than thepre-exercise value. By 48 hours after exercise, the change in MDA valuesreturned to baseline in this group. In the Vitamin E group the change inMDA values dropped from pre-exercise to post-exercise and returned tobaseline in the 24 and 48-hour post exercise samples. TABLE 3 Changes inPlasma MDA over time Change from Pre to 24 Pre to 48 Baseline Pre toPost hour post hour Post Placebo +0.326* +0.268*  +0.282* ATX 0.135−0.485* −0.004 Vitamin E −0.336* −0.071  −0.069*= Significantly different from baseline value within treatment group

Plasma F2-isoprostane (F2I).

Table 4 shows the values for plasma F2I. There were no significantincreases in plasma F2-isoprostane concentration between pre-exerciseand post-exercise samples within any of the treatment groups. Theplacebo group showed an increase in F2I at 24 hours post-exercise; by 48hours post-exercise the value had returned to the baseline value. F2Ivalues in the Astaxanthin group dropped below the baseline value forimmediate post-exercise, 24-hour post-exercise, and 48-hourpost-exercise measurements. In the Vitamin E group F2I values forpre-exercise, post-exercise, and 24-hour post-exercise were notsignificantly different from each other. The value for 48 hourspost-exercise was significantly lower than the other time periods forthe Vitamin E treatment group.

Between the three groups there were several significant differences.Compared to the placebo group, the Astaxanthin group had lower F2Ivalues immediately post-exercise and at 24 hours post-exercise. Thevalue of Vitamin E F2I was not different from the placebo value exceptat the 24 hour post-exercise time period where it was lower. The onlysignificant difference between Astaxanthin F2I values and Vitamin E F2Ivalues occurred immediately after exercise. The Astaxanthin value waslower than the Vitamin E value for this time period. TABLE 4 Plasma F2Isoprostane concentration (mean +/− se) Time Pre-ex Post-ex 24 hour post48 hour post Placebo 220 138 356 186 (+/−73) (+/−70) (+/−144)* (+/−110)ATX 167  52  90  66 (+/−111) (+/−13)*, a, b (+/−52)*, a (+/−21)* VitaminE 144 223 130  78 (+/−56) (+/−109) (+/−48) (+/−20)**= significantly different than baseline value within treatment groupa= significantly different from placebo within same time periodb= significantly different from Vitamin E treatment group within thesame time period

Plasma Total Oxidative Radical Absorptive Capacity (ORAC).

Table 5 shows the plasma values for total ORAC. In the placebo group,ORAC values increased from pre-exercise to post-exercise and remainedelevated through 24 hours post-exercise. Placebo ORAC values returned tobaseline values by 48 hours after exercise. There was no change in ORACbetween pre-exercise and post-exercise in the Astaxanthin group. ORACvalues fell below the baseline value in the 24-hour and 48-hour samplesin the Astaxanthin group. In the Vitamin E group, the ORAC values fellfrom pre-to-post exercise, and then rose back to the baseline value at24 hours post-exercise and then fell below baseline again at 48 hourspost-exercise. The changes in plasma total ORAC values within treatmentsacross time are similar to those obtained for MDA. TABLE 5 Plasma totalORAC (mean +/− se) Time Pre-ex Post-ex 24 hour post 48 hour post Placebo5430 7330 7790 4950 (+/−408)a (+/−408)* (+/−408)* (+/−408) ATX 7980 74605700 4800 (+/−361) (+/−361) (+/−361)*, a (+/−361) Vitamin E 7480 58507590 4840 (+/−390) (+/−390)*, a (+/−390) (+/−390)*= significantly different from baselinea= significantly different from other treatments for this same timeperiod

Plasma Creatine Kinase (CK).

Table 6 shows the plasma CK values. In all three groups the plasma CKvalues increased from pre-exercise to post-exercise and remainedelevated for all remaining time periods. The Astaxanthin group also hadlower CK values than the Vitamin E group for all time periods but 24hours post-exercise where there was no significant difference betweenthe two groups. None of the values measured in this experiment exceededthe normal range for CK. TABLE 6 Plasma Creatine Kinase (mean +/− se)Time Pre-ex Post-ex 24 hour post 48 hour post Placebo 134 176 220 186(+/−12) (+/−21)* (+/−50)* (+/−40)* ATX 109 152 171 158 (+/−11)a(+/−18)*, a (+/−24)* (+/−30)*, a Vitamin E 139 208 184 204 (+/−16)a(+/−16)* (+/−24)* (+/−25)**= significantly different from baseline within the treatment groupa= significantly different from Vitamin E treatment group

The Vitamin E-treated group maintained the greatest concentration ofVitamin E for each time period sampled. All groups showed an increase inplasma Vitamin E across time. This may be attributed to increased fatmobilization during exercise with concomitant incorporation of storedVitamin E into circulating lipoproteins. It has previously beendemonstrated that increases in plasma fatty acids and triglycerides indogs during low level prolonged exercise were concomitant with increasesin plasma Vitamin E. (Reynolds, A J, et al., J Nutr . 124(12Suppl):2754S-2759S (1994). While the placebo and Astaxanthin groupsmaintained Vitamin E concentrations above the low normal limitsestablished for dogs (below 60 would generally be considered somewhatlow), the drop in MDA observed during exercise in the Vitamin E treatedgroup suggests that there may be a benefit to supplementing Vitamin Eabove the baseline levels found in the diet.

The changes in MDA values suggest that Vitamin E supplementation mayhelp protect against oxidative damage during exercise while Astaxanthinsupplementation may protect against such damage during the first 24hours of recovery from exercise. These findings suggest that these twoantioxidants may have differing efficacies against different types ofoxidative stress. Vitamin E may be most effective against membrane lipidoxidation associated with exercise-induced elevations of oxygenmetabolism. Astaxanthin may be most effective against oxidative stresscaused by increased immune cell activity in the 24 hours followingexercise. These observations suggest a potential for synergism if thetwo antioxidants are combined.

Astaxanthin was found to protect the dogs from free radicals duringimmediate post exercise and up to 24 hours after exercise. Astaxanthinhelped protect against peroxidative damage as measured by F2I duringexercise and for the first 24 hours of recovery. Vitamin E treated dogsalso showed improvement over placebo fed dogs at 24 hours post-exercise.F2I measures lipid peroxidation from sources other than those measuredby MDA. The F2I data collected here suggests that Astaxanthin may betterprotect immune cells and sub cellular organelles during exercise thanVitamin E. Since F2I are byproducts of immune cells, the timing of theprotection of the Astaxanthin suggests that much of its effects aremediated through modulation of immune cell function.

Both Astaxanthin and Vitamin E groups showed a decrease in ORAC duringthe time periods when these treatments were most successful ininhibiting lipid peroxidation. One reason the Vitamin E and AstaxanthinORAC values dropped during exercise and at 24 hours post-exercise,respectively, may be that the supplemented antioxidants may have beenconsumed to contain lipid peroxidation during these time periods.

The fact that CK values were the lowest in the Astaxanthin group fornearly all measurements suggests that this supplement is protecting themuscle cell membranes from damage during exercise better than eitherVitamin E or the placebo. The exercise in this study did not induceelevations in plasma CK above the normal range.

Compared to the placebo group, both Vitamin E and astaxanthin treatmentshowed improved protection against the oxidative stress associated withexercise. Vitamin E appears to be most effective against malondialdehydeproduction during exercise and most effective against F-2 isoprostaneproduction during the first 24 hours after exercise. Astaxanthinsupplementation was better at decreasing MDA production during the first24 hours after exercise and better at curbing isoprostane productionduring exercise and the first 24 hours of recovery. These findingssuggest that Vitamin E and Astaxanthin act in different cellularcompartments and or pathways from each other. They also indicate apotential for at least an additive effect, and possibly a synergisticeffect, if the two are combined.

Vitamin E supplementation resulted in decreased malondialdehydeconcentrations during exercise when compared to the placebo group.Astaxanthin supplementation was associated with a decrease inmalondialdehydes 24 hours post-exercise when compared to Vitamin E andplacebo groups. ORAC (Oxygen Radical Absorption Capacity, which is anindex of total anti-oxidant status) was not improved by either Vitamin Eor astaxanthin supplementation for any of the time periods measured.Creatine kinase activity (an index of muscle cell membrane damage) andF-2 isoprostane (a measure of lipid oxidation from prostaglandins)concentrations only showed significant differences in the astaxanthintreated group and only in the immediate post-exercise and 24 hourpost-exercise samples where they were lower than values measured ineither Vitamin E or placebo groups. These findings indicate that bothastaxanthin and Vitamin E show some protective effect against oxidativedamage in exercising dogs. Astaxanthin showed a broader range ofprotection across a greater time period than Vitamin E. The resultsdescribed herein suggest that a combination of Vitamin E and astaxanthinmay have an effect that is at least additive and may be synergistic. Itis thus recommended that astaxanthin be included in diets intended fordogs that may experience oxidative stressors including, but not limitedto, exercise, infection, or trauma.

Example 2

Animals are administered a composition comprising a combination ofastaxanthin and Vitamin E (alpha-tocopherol) to determine the effect onoxidative stress due to exercise.

Dogs are maintained on the same basal diet (Purina Pro Plan® Performancechicken and rice formula) for one month prior to the onset and theentire duration of the treatment. Two groups of dogs are fedcommercially available Pro Plan® Performance chicken and rice formuladog food as a basal diet, and each group is fed either: a placeboconsisting of about 0.5 g maltodextrin, or a combination of about 2 mgastaxanthin and about 400 mg Vitamin E (alpha-tocopherol). Pro Plan® dogfood is available from Nestle Purina Pet Care Company in St. Louis, Mo.The dogs are trained three times a week on an exercise wheel for aperiod of six weeks. On the sixth week, the dogs rest for four days andthen exercise three days in a row. Blood samples are collected beforeexercise on day one, immediately after exercise on day three, and at 24hours and 48 hours after exercise. Plasma from the blood samples isanalyzed for concentrations of malondialdehyde, F-2 isoprostanes,Vitamin E, creatine kinase activity as well as oxygen radical absorptioncapacity (ORAC). Decreases in measures of oxidative stress and/orincreases in measures of antioxidant status is indicative ofanti-oxidative effects of a given treatment.

Example 3

An in vitro experiment may be performed using canine lens epithelialcells supplemented with astaxanthin at physiological levels. The studieswould demonstrate that astaxanthin can significantly reduce free radicalformation and lipid peroxidation products in canine lens epithelia whenstressed with ultraviolet (UV) light. Additionally, when the astaxanthinsupplemented canine lens epithelial cells are stressed with UV light,there would be significantly less cell death. The results would indicatethat a decrease in free radical formation and lipid peroxidationproducts along with the decrease in cell death show that astaxanthinsupplementation reduces the risk or severity of cataract formation indogs.

Example 4

An in vivo study could also be performed to demonstrate the beneficialeffects of astaxanthin in combating cataract formation. In a modelstudy, 40 dogs could be chosen and randomly allocated to either thecontrol group or to a treatment group receiving AAFCO recommended levelsof antioxidants. The treatment group would receive astaxanthin and mayalso receive one or more of Vitamin E, lutein, zeaxanthin and zinc. Atthe conclusion of the treatment times (a two year study, for example),it would be shown that the control group has significantly more cataractformation and the severity of cataract would be significantly greater asassessed by a slit-lamp test.

Example 5

In vitro experiments may be performed to demonstrate that astaxanthinimproves or enhances immune system function. In vitro assays to assessimmune function are well-known to those of skill in the art. Manyprotocols are known and may be used to perform such assessments ofimmune function. Examples of such protocols are available in variousjournal articles and texts (e.g., Current Protocols in Immunology, JohnE. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach,Warren Strober, eds. John Wiley & Sons, NY, 1999).

To assess the effect of astaxanthin supplementation on immune function,dogs may be fed a diet containing astaxanthin and/or Vitamin E for 16weeks. The amount of astaxanthin used may be 0, 1, 5, 10, 20, and 40 mgper animal per day. Various assays may be performed to assess immunefunction.

In one assay, for example, peripheral blood mononuclear cells (PBMCs)may be stimulated in vitro with a polyclonal antisera, or Concanavalin A(ConA), phytohemagglutinin (PHA) or pokeweed mitogens.Lymphoproliferation is assessed by any known method.

In another in vitro lymphoproliferation test, astaxanthin may be addeddirectly to the media of PBMC culture during stimulation to demonstratethat astaxanthin increases proliferation of cells significantly morethan the proliferation observed in the absence of astaxanthin.

In another assay, natural killer (NK) cell cytotoxicity is assessed forNK cells isolated from animals that are fed a diet including astaxanthinsupplementation. The NK cell assay would demonstrate that NKcytotoxicity is significantly increased in animals fed a diet containingastaxanthin as opposed to control animals not fed astaxanthin.

Delayed-type hypersensitivity may also be assessed in animals given adiet supplemented with astaxanthin. In these assays, control animals andanimals fed a diet containing astaxanthin are given cutaneous injectionsof saline, a mitogen or a vaccine of distemper virus. The animals arethen assessed to measure the skin reactions to mitogen, virus andcontrol. In animals fed a diet containing astaxanthin, the skinreactions are more robust for virus and mitogen than that found incontrol animals.

Control animals and animals fed a diet containing astaxanthin may alsobe assessed for the robustness of the immune response against anantigen. For example, antibody titers against an antigen may be measuredfor control animals and animals fed a diet containing astaxanthin.Animals fed a diet containing astaxanthin would have higher antibodytiters than control animals.

Example 6

Osteoarthritis (OA) is also associated with oxidative stress. OA ischaracterized by damage to the articular cartilage. This damage ismediated by enzymes (MMPs) that are specific for collagen, elastin andgelatin. Upregulation of these enzymes has been shown to be caused byfree radicals including, but not limited to nitric oxide, superoxideanion, and hydrogen peroxide.

A diet supplemented with astaxanthin may be shown to reduce inflammationand associated pain in osteoarthritis. A study to demonstrate thebeneficial effects of astaxanthin in the diet could be performed byrandomly assigning a group of 24 dogs to either a control group or agroup fed a diet containing astaxanthin at 1, 5, 10, 20, or 40 mg peranimal per day. At 12 weeks of treatment, plasma and synovial fluidsamples are taken from each animal and assayed for total antioxidantactivity, and levels of malionaldehyde, prostaglandin, isoprostanes,MMPs and TIMPs. Additionally, the gait of the dogs may be assessed usingstandard force plate analyses. Biochemical analyses would show thatastaxanthin supplementation results in a decrease in isoprostanes, totalantioxidant activity and MMPs (in both plasma and synovial fluid.Treated dogs would show significant improvement in gait when compared tocontrols. The experiment would demonstrate that astaxanthin improves ordelays the progression of OA in dogs.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

Without departing from the spirit and scope of this invention, one ofordinary skill can make various changes and modifications to theinvention to adapt it to various usages and conditions, mutatismutandis. As such, these changes and modifications are properly,equitably, and intended to be, within the full range of equivalence ofthe following claims.

1. A composition comprising astaxanthin, wherein the composition isadapted for use by a companion animal.
 2. The composition according toclaim 1 comprising less than about 3% of astaxanthin, by weight of thecomposition.
 3. The composition according to claim 2 wherein thecomposition is a nutritionally balanced pet food composition.
 4. Thecomposition according to claim 3 comprising from about 0.0001% to about2% of astaxanthin, by weight of the composition.
 5. The compositionaccording to claim 4 which is selected from the group consisting of dogfood compositions, cat food compositions, and combinations thereof. 6.The composition according to claim 5 comprising from about 0.001% toabout 1% of astaxanthin, by weight of the composition.
 7. Thecomposition according to claim 6 comprising from about 0.001% to about0.5% of astaxanthin, by weight of the composition.
 8. The compositionaccording to claim 2 wherein the composition is a supplement.
 9. Thecomposition according to claim 8 comprising from about 0.0001% to about2% of astaxanthin, by weight of the composition.
 10. A method selectedfrom the group consisting of attenuating inflammation, enhancingimmunity, enhancing longevity, and combinations thereof, comprisingadministering to a companion animal a composition comprising aneffective amount of astaxanthin.
 11. The method according to claim 10comprising administering to the companion animal from about 0.001 mg toabout 40 mg, daily, of astaxanthin to the companion animal.
 12. Themethod according to claim 11 wherein the immune response is acell-mediated immune response.
 13. The method according to claim 11wherein the immune response is a humoral immune response.
 14. The methodaccording to claim 11 wherein the companion animal is a domestic dog.15. The method according to claim 14 wherein from about I mg to about 40mg, daily, of astaxanthin is administered to the domestic dog.
 16. Themethod according to claim 11 wherein the companion animal is a domesticcat.
 17. The method according to claim 16 wherein from about 0.001 mg toabout 10 mg, daily, of astaxanthin is administered to the domestic cat.18. A composition comprising astaxanthin, the composition beingeffective for reduction or prevention of oxidative stress in an animal.19. The composition of claim 18, formulated to deliver the astaxanthinin an amount of about 0.001 to about 40 mg per day to the animal. 20.The composition of claim 19, formulated to deliver the astaxanthin in anamount of about 0.1 to about 8 mg per day to the animal.
 21. Thecomposition of claim 18 further comprising Vitamin E.
 22. Thecomposition of claim 21, formulated to deliver about 15 to about 500 mgVitamin E per day to the animal.
 23. The composition of claim 18,wherein the animal is a mammal.
 24. The composition of claim 23, whereinthe animal is a dog.
 25. The composition of claim 23, wherein the animalis a cat.
 26. A method for reducing or preventing oxidative stress in ananimal, the method comprising administering to the animal a compositioncomprising astaxanthin in an amount effective to reduce the oxidativestress in the animal.
 27. The method of claim 26 wherein the compositionfurther comprises Vitamin E.
 28. A method of promoting exercise recoveryin an animal comprising administering to the animal a compositioncomprising astaxanthin in an amount effective to promote the exerciserecovery in the animal.
 29. The method of claim 28 wherein thecomposition further comprises Vitamin E.
 30. A method for reducing theamount of an oxidative stress-associated molecule in an animalcomprising administering an effective amount of astaxanthin to theanimal wherein the astaxanthin is effective in reducing the level of atleast one oxidative stress-associated molecule selected from the groupconsisting of nitric oxide, malondialdehyde, F2 isoprostanes, lipidperoxidation products, creatine kinase, superoxide anion and hydrogenperoxide.
 31. The method of claim 30 further comprising administering aneffective amount of Vitamin E.
 32. The method of claim 30, wherein thereduction in the at least one oxidative stress-associated molecules inthe animal results in reduction, prevention or delay in onset of adetrimental physiological condition associated with oxidative stress.33. The method of claim 32, wherein the detrimental physiologicalcondition is inflammation, cataract formation, osteoarthritis, reducedlongevity or reduced immunity.